Process for preparing pyrophosphate nucleotides



United States Patent Office 35,188,310 Patented June 8, 19 65 3,138,310 PROCESS FOR PREPARING PYROPHGSPHATE NUCLEOTIDES Teruaki Mulraiyama, Tokyo, and Tsujiaki Hata, Yokosulra-shi, Japan, assignors to Asahi Kasei Kogyo giabnshiki Kaisha, Osaka, Japan, a corporation of apan No Drawing. Filed Ap 1, 1963, Ser. No. 269,713 Claims priority, application Japan, Mar. 31, 1962, 37/12,129 3 Claims. (Cl. 260-2115) This invention relates to processes for preparing pyrophosphates.

The invention is concerned primarily with processes for preparing pyrophosphates from phosphates and phos phites in the presence of a-monoor di-halogen derivatives of active methylene compounds.

The process of this invention follows the following general equation:

11 o \ORA Wherein and R is hydrogen or alkyl, aralkyl or aryl radicals.

As will be apparent from the above formula, the process of this invention includes not only the preparation of nucleoside poly-phosphates such as adenosine dior triphosphate, uridine dior tri-phosphate, carbohydrates polyphosphates which are important biochemically and pharmaceutically, but also the preparation of several phosphorylating agents such as tetraethyl pyrophosphate or tetra(p-nitrophenyl)pyrophosphate.

For the preparation of pyrophosphates from phosphates, various attempts have been made by using carbodiimides, ketoXime-sulfonates, isocyanates, phosphoramidates. These methods, however, have much difiiculty in being applied commercially, because they involve, complex processes and the diflicult removal of by-products. By these methods, only symmetrical pyrophosphates are prepared.

It has been also well known that the enolphosphates formed from trialkyl phosphites and a-haloketones or -u-haloaldehydes by the Perkow reaction does not further react with the trialkyl phosphites but reacts exclusively with nucleophilic phosphates to make pyrophosphates. Any of the enol-phosphates obtained by the Perkow reaction up to the present time, however, has not been applied industrially to prepare pyrophosphates because they are prepared under severe conditions and are sluggish toward nucleophilic reagents (I. F. Allen and O. H. Johnson, J. Am. Chem. Soc., 77, 2871 (1955)). The inventors have recently found that the enol-phosphate prepared from triethyl phosphite and ethyl a-chloroacetone has slightreactivity toward monoor di-ethyl phosphate and make tetraethylpyrophosphate (55% yield) under the presence of a catalytic amount of boron fluoride at C. for 1 hour. The enol-phosphate, however, does not have enough reactivity to make pyrophosphates from other less reactive phosphates.

This invention is primarily concerned with commercial methods to prepare the pyrophosphates of naturally occurring compounds such as carbohydrates, nucleosides, vitamins, etc., which are important biochemically and pharmaceutically.

This invention concerns the processes for the preparation of various pyrophosphates in quantitative yield by reacting phosphates.

R2 OH O in the presence of monoor di-halogen derivatives of active methylene compounds and to prepare then pyrophosphates (R O\ o 0\ /OR5) P P R O o om quantitatively. The chemical reaction of this invention is assumed to proceed as follows:

ACHXCOY ACH=CY ll RO-P-OR I H R OPOR RO-POR3 ACH2COY R 0 .0 0 0R5 R 0 0 0B The reaction proceeds through the initial formation of the enol-phosphates by the interaction of monoor di halogen derivatives of active methylene compounds with phosphites followed by the attack of nucleophiiic phos phates to form pyrophosphates.

As the above phosphites in this invention, trialkyl phosphites consisting of methyl, ethyl, nor iso-propyl or nor iso-butyl, or tribenzyl phosphite are generally used, because they are inexpensive. In some purpose, the phosphites substituted by different groups such as monoethyl bis(p-nitrophenyl) phosphite are used.

As the halogen derivatives of active methylene com pounds in this invention, any of the halogens may be employed. Generally, bromine or chlorine substituents of cyanoacetamide malonamide, alkyl cyanoa-cetate.

(wherein alkyl is C -C or N-cyclohexyl cyanoa'cetamide are suitable.

As the phosphates, monoor di-alkyl phosphates consisting of methyl, ethyl, nor iso-propyl, or nor isobutyl, aralkyl phosphates such as monoor di-benzyl phosphate, aryl phosphates, suchas monoor di-phenyl, monoor bis-p-nitrophenyl, monoor di-pchlorophenyl phosphate, carbohydrate'phosphates such as glucose, fructose, ribose phosphates, nucleoside phosphates such as 5'-guany-lic acid, 5adenylic acid, 3'-uridylic acid, etc., or the phosphates of ster-ols, terpene alcohols or vitamins are used.

As will be apparent from the general formula, when .a suitable period at room temperature.

phosphites and phosphates have the same radicals, symmetrical pyrophosphates are obtained (R =R =R =R phate is devived from 5'-adenosine monophosphate by pyrophosphonylating twice with some of the phosphites such as dibenzyl or diphenyl phosphites followed by hydrogenolysis.

In carrying out this invention, generally, one mole of the above phosphites and one mole of the above phosphates are dissolved in the solvent in different containers, and to either of which one or more kinds of the above halogen derivatives of the active methylene compounds is added in equivalent amount to halogen. These solutions are mixed at a suitable temperature and kept for After filtering 01? a white precipitate which is an active methylene compound, the pyrophosphates are collected by distilla tion, solvent extraction or their combination.

The active methylene compounds filtered off are recovered at 90-95% yield and re-halogenated for subsequent use for the same purpose.

The reaction condition such as the reaction time and the reaction temperature required for this reaction is a function of reactants, a period of 1-2 hours at room temperature being required when the reactive reactants such as methyl, ethyl or phenyl phosphate are employed. When less reactive reactants such as the phosphates of carbohydrates, nucleosides or vitamins are employed, however, a period of 24-48 hours at room temperature is suitable.

For carrying out this invention, any solvent. which is inactive to the enol-phosphates formed by the Perkow reaction may be used. Generally, a solvent easily dissolving the reactant such as ether, benzene, dioxame, N-dimethyl formamide and tetrahydrofuran are employed. It is not necessary to dry these organic solvents before using, because water does not inhibit this reaction. Sometimes, the reaction can be ettected in aqueous solution.

Most of the halogen substituents of active methylene compounds employed in this invention shown as the general formula ACHXCOY, ACX COY are superior to ethyl monobromomalonate in the following respects.

(1) The Perkow reaction occurs more easily (2) The enol-phosphate formed are more reactive to nucleophilic phosphates Furthermore, this invention has the advantage of preparing pyrophosphate by a one step process by reacting phosphates with phosphites in the presence of the halogen derivatives of active methylene compounds without isolating the intermediate enol-phosphate as compared for example to the method reported by Cramer and Goiirtener, Chem. Ber., 91 7041, 1958, in which the 'enol-phosphate was isolated.

Compared with other condensation or dehydration methods, the present invention is preferable, because more easily produced phosphites are used and recovery and cyclization of the halogen derivatives of active methylene compounds can be effected more effectively.

In addition to the above, the invention can be applied to prepare unsymmetrical pyrophosphates in which most of the pyrophosphates are of naturally occurring compounds.

The following examples are illustrative of the invention:

4.- Example 1 To 1.54 grams of diethyl phosphate and 1.63 grams of a-monobromocyanoacetamide in 150 ml. of ether at -50 C. were added dropwise 1.24 grams of trimethyl phosphite dissolved in 15 ml. of ether. After standing for 2 hours at room temperature, a white precipitate was filtered and ether and methylbromide are removed under vacuum. 2.4 grams (yield 92%) of dimethyl diethyl pyrophosphate were obtained by distillation (B.P. 106 C./0.004 mm.).

Example 2 From 1.54 grams of diethyl phosphate, 1.22 grams of a-di-bromocyanoacetamide and 1.66 grams of triethyl phosphite, 2.7 grams (yield 94%, B.P. -136 C./1 mm.) of tetraethyl pyrophosphate were obtained by treating exactly as Example 1.

Example 3 From 1.54 grams of diethylphosphate, 1.2 grams of a-monochlorocyanoacetamide and 2.5 grams of tri-n-butyl phosphite, 3.3 grams (yield 96%, B.P. 114-118 C./0.02 mm.) of diethyl di-n-butyl pyrophosphate were obtained by treating exactly as in Example 1.

Example 4 The reaction of 1.26 grams of dimethyl phosphate with 1.3 grams of dibromomalonamide and 1.66 grams of triethylphosphite in ether was made at room temperature for 2 hours. Treatment of the reaction mixture exactly as in Example 1 produced 2.3 grams (yield 88%) of dimethyl diethyl pyrophosphate.

Example 5 The reaction of 1.02 grams of bis-(p-nitrophenyl)phosphate with 3.9 grams of dibromomalonamide and monoethyl bis-(p-nitrophenyl)phosphite in dioxane was conducted at room temperature for 5 hours. After white precipitate was filtered, removal of dioxane and ethyl bromide under vacuum gave crude tetra- (p-nitrophenyl)- pyrophosphate. Without purification, this product was used as a phosphorylating agent of 2',3-o-isopropylidene guanosine to make guanosine-5'-phosphate.

Example 6 Treatment of 1.54 grams of diethyl phosphate with 1.66 grams of triethylphosphite and any one of the mixture of the following compounds:

0.77 gram of a-dichlorocyanoacetamide 2.10 grams of u-monoiodocyanoacetamide, 1.92 grams of ethyl oc-HIODO- bromocyanoacetate, exactly as in Example 1 produced 1.9-2.3 grams (yield 66-88%) of tetraethyl pyrophosphate.

Example 7 To a mixture of 0.35 gram of 5'-adenosine monophosphate, 0.66 gram of tribenzyl phosphite and 0.37 gram of n-butylamine dissolved in 25 ml. of N-dimethyl formamide were dropwise added 0.25 gram of a-monobromocyanoacetamide dissolved in 10 ml. of N-dimethyl formamide. The reaction mixture was stirred for 6 hours, then stood for additional 24 hours at room temperature.

' After adding 0.25 gram of sodium iodide, the reaction mixture was stirred again for 2 hours.

To this solution kept at 40 C., 100 ml. of dry acetone were added, then a precipitate was collected by centrifuge action, washed with acetone and dried over phosphorus entaoxide to give 0.4 gram of dry solid.. After extracting this solid 15 times with 15 ml. each of isopropyl a1- cohol, removal of solvents gave 0.30 gram (yield 60.7%) of monobenzyl 5'-adenosine diphosphate.

This monobenzyl 5-adenosine diphosphate was derived to 5-adenosine triphosphate which is important biochemically and pharmaceutically.

What we claim is:

1. In a process wherein a phosphite having the formula:

in which R R R are selected from the group consisting of benzyl, p-nitrophenyl and alkyl having 1 to 4 carhon atoms, is added to a phosphate selected from the group consisting of monoalkyl phosphate, 'dialkyl phosphate, monobenzyl phosphate, dibenzyl phosphate, monophenyl phosphate, diphenyl phosphate, p-nitrophenyl phosphate, bis-paranitrophenyl phosphate, chlorophenyl phosphate, dichlorophenyl phosphate, and a nucleotide, said alkyl having 1 to 4 carbon atoms to produce the corresponding pyrophosphates, an improvement which comprises conducting the addition in the presence of at least one active methylene compound selected from the group consisting of chloro and bromo derivates of N- cyclohexyl cyanoacetamide, cyanoacetamide, malonamide, and alkyl cyanoacetate which alkyl 'has 1 to 4 carbon atoms, and a solvent for the phosphite and the active methylene compound, said solvent being nonreactive to enolphosphates formed by the Perkow reaction.

2. In a process wherein a phosphite having the formula:

6 in which R R and R are selected from the group consisting of an alkyl having 1 to 4 carbon atoms, benzyl and p-nitrophenyl, and a member selected from the group consisting of monoalkyl phosphate, dialkyl phosphate, monobenzyl phosphate, dibenzyl phosphate, monophenyl phosphate, diphenyl phosphate, p-nitrophenyl phosphate, bis-paranitrophenyl phosphate, chlorophenyl phosphate, dichlorophenyl phosphate are added together to produce the corresponding pyrophosphate, said alkyl being selected from the group consisting of methyl, ethyl, npropyl, isopropyl, n-butyl and isobutyl, an improvement which comprises the addition being efiected at a temperature of about C. for 2-3 hours in the presence of at least one active methylene compound selected from the group consisting of chloro and bromo derivates of cyanoacetamide, malonamide, N-cyclohexyl cyanoacetamide, and alkyl cyanoacetate which alkyl has 1 to 4 carbon atoms.

3. In a process wherein a phosphite having the formula:

derivative of an active methylene compound having substituted bromo and chloro atoms selected from the group consisting of cyanoacetamide, malonamide, and N-cyclohexyl cyanoacetate which alkyl has 1 to 4 carbon atoms.

References Cited by the Examiner Cramer: Angew. Chem. vol. 72, ,April 1960, pp.

LEWIS GOTTS, Primary Examiner. 

1. IN A PROCESS WHEREIN A PHOSPHITE HAVING THE FORMULA: 